Recently, DNA vaccines have been developed for diseases like influenza, hepatitis B, HIV and cancer. DNA vaccines, like the cancer associated antigen carcinoembryonic antigen (CEA), have high protection rates against in vivo challenge with a human breast cancer cell line. However, potential dangers involved with DNA vaccines has lead to investigating the feasibility of RNA vaccines. Detectable immune responses have been produced in mice immunized with mRNA encoding influenza virus nucleoprotein and CEA. These results warrant continued research. The 5' cap of mRNA is important in promoting the stability and efficient translation of mRNA. Thus, RNA vaccines should be composed of 100% capped transcripts. Currently, the best method for synthesizing capped RNA is unsatisfactory in that it produces a mixture of "forward" and "reverse" capped RNA and the yields are much lower compared to the synthesis of uncapped RNA. Our main goal is to develop an efficient method for manufacturing a potent RNA vaccine suitable for use in humans. We will approach this goal by developing methods and reagents to: 1) optimize production of "forward" capped RNA; 2) purify full- length, capped RNA; and 3) increase the nuclease resistance of the capped RNA in order to enhance protein expression. PROPOSED COMMERCIAL APPLICATION: The long term goal of this research is to produce human therapeutic RNA, especially for cancer vaccines but also including anti-sense RNA and ribozymes. In the short term, this research may lead to the development of inexpensive kits, commercially available for synthesizing capped RNA and for synthesizing nuclease resistant RNA.